1. Field of the Invention
The present invention relates to a method for producing lactosyl glycosides or N-acetyllactosaminyl glycosides. More specifically, the present invention relates to a method for producing lactosyl glycosides or N-acetyllactosaminyl glycosides, using an enzyme having an activity of cleaving a xcex2-1,4 glucosyl bond.
2. Description of the Prior Art
Oligosaccharides and glycoconjugates (glycoproteins, glycolipids, glycosides, etc.) are present in animals and plants, for example, biological cells, body fluids, fruits, seeds, etc. These components have essential functions to maintain or regulate biological functions such as the transmission of biological information on the surface of a cell membrane, the morphogenesis of cells, the maintenance of a specific structure that is composed of covalent bonds with proteins, just like genes and proteins.
Glycolipids, certain kinds of glycoconjugates, are classified into sphingoglycolipids, glycoglycerolipids and others. Sphingoglycolipids are present mostly in animal tissues, and are classified roughly into four series, namely the globo-, the lacto, the ganglion, and the gala-series, depending on their basic sugar chain component. Three of those series, the globo-, the lacto-, and the ganglio-series, have a lactose unit and lactosylceramide unit in which lactose is bound to ceramide as the basic backbone, and are classified depending on the saccharide to be added next.
Lactosylceramide has a Galxcex21-4Glc unit, that is, a lactose unit. It has come to be known that bacteria recognize this lactose unit to recognize the sequence of a glycoconjugate. The mechanism that a microorganism adheres to a host cell via a sugar chain plays an important role as a model of inter-cell recognition. Furthermore, it has come to be known that bacteria not only recognize the end of a sugar chain on the surface of a cell for binding, but also recognize the sugar chain structure inside the sugar chain. For example, it is known that E. coli, microorganisms belonging to genus Pseudomonas, Actinomycetes, and propionibacteria recognize the lactose unit that is present inside of various sugar chains as well.
Thus, lactosylceramide as described above plays a physiologically important role in such as adhesion of cells. Therefore, lactosyl glycoside can be used for the applications of medicine, food and the like, but it is not easy to produce the lactosyl glycoside at present.
There are many kinds of glycosides as glycoconjugates. For example, the glycosides which are composed of sugar unit (glucose, galactose and N-acetyllactosaminide), and glycerol as the aglycon, are known. Naturally occurring components such as glyceroyl glucoside (2-O-xcex1-D-Glucosylglycerol, (2R)-1-O-xcex1-D-Glucosylglycerol, and (2S)-1-O-xcex1-D-Glucosylglycerol) are known as the glycoside for glucose. Glyceroyl galactoside (1-O-xcex2-D-Galactosylglycerol) as a chemically synthesized substance is known as the glycoside for galactose. As the glycoside for N-acetyllactosaminide, the existence of O-xcex2-D-Galacto-pyranosyl-(1xe2x86x924)-O-xcex2-D-2-Acetyl-amino-2-deoxyl-Glucopyranosyl-(1xe2x86x922)-Gycerol is reported.
Among the abovedescribed glycosides, glyceroyl glucoside, glyceroyl galactoside and the like are characterized by their taste, their moisture retaining properties, and their physiological functions. These substances are also useful as a starting material for the synthesis of glycoglycerolipids. Glycoglycerolipids are present widely in organisms, ranging from microorganisms to higher plants, and are reported to have emulsification stability and carcinogenic promoter suppressing effects. Therefore, compounds in which saccharides having different structures are added to glycerol can be expected to have new functions or to be applied as new starting materials of glycoglycerolipids.
N-Acetyllactosaminyl glycosides are known to be present in a receptor recognized by Staphylococcus or the core unit of the sugar chain structure recognized by influenza viruses A and B, Sendai virus, Newcastle disease virus and the like, and participate in the recognition of cells. Therefore, the N-acetyllactosaminyl glycosides are useful for medical applications, but it is not easy to produce N-acetyllactosaminyl glycosides at present.
The functions of such saccharide-related substances are noted, therefore, modification and substitution research (remodeling) of sugar chains and glycoconjugates is performed in order to utilize the substances for medical and food applications by improving these functions further or modifying their physiological functions.
For remodeling the sugar chains of saccharides and glycoconjugates, there are chemical methods, enzymatic methods, and methods combining chemical and enzymatic methods.
As a chemical method, there is an organic synthesis method for sugar chain synthesis, but this method involves many complicated steps, and a process for removing byproducts (purifying a desired substance) is necessary. Furthermore, reagents that are harmful to human health are often used, and there is the fear that the waste liquid may damage the surrounding environment.
On the other hand, for remodeling sugar chains enzymatically, a method using transferase, exoglucosidase, endoglucosidase, and the like are known.
One of the methods using transferase or exoglucosidase is described in D. H. Joziasse et al. Eur. J. Biochem., vol. 191, pages 75-83 (1990), which discloses a method of sequentially adding a glucosyl group to a nonreducing end of a sugar chain by using exoglucosidase or glucosyltransferase.
However, although the sugar chain synthesis using exoglucosidase or glucosyltransferase is easier than chemical synthesis, it is still necessary to perform an enzyme reaction sequentially for each saccharide residue one by one, so that a large number of reaction steps are required, which makes the method troublesome.
Examples of the disclosure of transglycosylation using endoglucosidase are as follows. R. B. Trimble et al. J. Biol. Chem., vol. 261, pp. 12000-12005 (1986) describe a method of using endo-xcex2-N-acetylglucosaminidase derived from Flavobacterium meningosepticum. R. M. Bardales et al., J. Biol. Chem., vol. 264, pp. 19893-19897 (1989) describe a method of using endo-xcex1-N-acetylgalactosaminidase derived from Diprococcus pneumoniae. Japanese Laid-Open Patent Publication (Tokkai) No. 5-64594 describes a transfer reaction of high-mannose type of sugar chains to saccharides by using endo-xcex2-N-acetylglucosaminidase derived from Arthrobacter protophormiae. K. Yamamoto et al., Biochem. Biophys. Res. Commun., vol. 203, pp. 244-252 (1994) describe a transfer reaction of a sugar chain to a saccharide by using endo-xcex2-acetylglucosaminidase derived from Mucor hiemalis. Japanese Laid-Open Patent Publication (Tokkai) No. 10-245402 describes a transfer reaction of (Man)6-GlcNAc using endo-xcex2-N-acetylglucosaminidase. Japanese Laid-Open Patent Publication (Tokkai) No. 10-33194 describes a transfer reaction of Galxcex21-3GlcNAc using lacto-N-biosidase.
As described above, examples of transferring various sugar chains by using endoglucosidases have been reported, but there has been no example of transferring lactose or N-acetyllactosaminide unit so far.
Therefore, there is a need for a method for producing lactosyl glycosides or N-acetyllactosaminyl glycosides in a simple manner.
The present invention is directed to a method for producing a lactosyl glycoside or an N-acetyllactosaminyl glycoside comprising using a transfer reaction represented by the following formula in the presence of an enzyme having an activity of cleaving a xcex21,4 glucosyl bond:
LacA-X+Yxe2x86x92LacA-Y+X,
wherein LacA represents lactose or N-acetyllactosaminide; X represents hydrogen (H), a saccharide, a glycoconjugate, or a phenolic compound; and Y represents a compound having an alcoholic hydroxyl group or a phenolic hydroxyl group.
In one preferable embodiment, Y is a compound having an alcoholic hydroxyl group.
In another preferable embodiment, the compound having an alcoholic hydroxyl group is an aliphatic alcohol or a saccharide.
In yet another preferable embodiment, the compound having an alcoholic hydroxyl group is an amino acid, a peptide or a protein having a serine or threonine residue.
In one preferable embodiment, X is hydrogen.
In one preferable embodiment, the enzyme having an activity of cleaving a xcex2-1,4 glucosyl bond is exo-cellobiohydrolase, xcex2-D-glucosidase, and/or cellulase.
The present invention is also directed to glyceroyl N-acetyllactosaminide (O-xcex2-D-Galactopyranosyl-(1xe2x86x924)-O-xcex2-D-2-Acetyl-amino-2-deoxyl-Glucopyranosyl-(1xe2x86x921)-Glycerol) represented by the structural formula (I): 